Edible mushrooms are cultivated throughout the world. Although the American consumer is most familiar with the white button mushroom (Agaricus bisporus), many other types of mushrooms are also cultivated and are often more popular than Agaricus. These non-Agaricus mushrooms are often collectively referred to as "specialty" or "exotic" mushrooms. Table 1 lists many, but not necessarily all, specialty mushroom types currently or potentially grown commercially. In some cases, "potentially grown" reflects no more than a desire to cultivate the mushroom.
TABLE 1 ______________________________________ Specialty mushrooms currently or potentially grown commercially ______________________________________ Agaricus bitorquis Agrocybe aegerita Amanita spp. Armillaria mellea Auricularia spp. Boletus spp. Cantharellus cibarius Collybia fusipes Coprinus spp. Flammulina velutipes Ganoderma lucidum Grifola frondosa Hericium erinaceus Hydnum repandum Hypsizygus marmoreus Kuehneromyces mutabilis Lactarius spp. Lentinula edodes Lepiota spp. Lyophyllum georgii Marasmius oreades Morchella spp. Pleurotus spp. Pholiota spp. Plicaria muralis Psalliota spp. Rhodopaxillus spp. Russula virescens Stropharia rugoso annulata Tremella fuciformis Trichloma matsutake Tuber spp. Volvariella spp. Peziza aurantia ______________________________________
The commercial production of mushrooms involves a series of steps, with the specific details and sequence of steps depending on the genus and species being cultivated. Details of specialty mushroom cultivation methods are given in many publications, including for example Chang & Hayes, 1976; Stamets & Chilton, 1983; Chang & Miles, 1989; Royse, 1997. Many specialty mushroom cultivation systems employ sawdust, straw, or waste wood substrates. An example of specialty mushroom production methods is found with the Shiitake mushroom (Lentinula edodes).
The traditional production method for Shiitake involves cultivation on natural hardwood logs. Freshly cut or overwintered logs are cut to convenient lengths, and holes are drilled at various locations in the logs. Wood spawn plugs are inserted in the holes and sealed with paraffin, and logs are incubated for 6 to 9 months. Wood spawn plugs consist of hardwood dowels that are moistened, sterilized, and inoculated with pure cultures of Leninula edodes. Following complete colonization of the plugs by the mycelium, they are ready for use. Alternately, the holes are packed with sawdust spawn (see below) and sealed with paraffin. Induction of mushroom production occurs by soaking the logs in water or mechanical agitation of the logs. Mushrooms are formed from primordia on the surface of the logs, and fruiting can continue for months or years.
A more modern and efficient method of Shiitake mushroom production is on "synthetic" or "sawdust" logs. In this technology, hardwood sawdust is mixed with additional nutrients (wheat bran, rice bran, millet, rye, corn, etc.), adjusted to ca. 60% moisture content, filled into autoclavable plastic bags, and subjected to steam sterilization. Once cooled, the substrate is inoculated with grain or sawdust Shiitake spawn (see below) and incubated to allow thorough colonization of the substrate with the Lentinula mycelium. During the growth process, the Lentinula mycelium knits the substrate together to form a firm block structure. Mushroom production is initiated by removing the bags, watering, mechanically agitating, illuminating, or other treatment. Mushrooms can be produced as soon as ca. 40 days after the initiation of the process. The use of the synthetic logs is substantially more efficient than natural logs in terms of space utilization, and total mushroom yields are significantly higher. Shiitake mushrooms have also been produced on a composted substrate filled into large wooden trays (not unlike the Agaricus production process, U.S. Pat. No. 4,874,419), but this method has not gained wide acceptance.
The cultivation of most other specialty mushroom types generally represent variations on the methods of cultivation of Shiitake on sawdust blocks. For example, Auricukiha spp. is grown on a sterilized substrate of sawdust, cottonseed hulls, bran, and/or cereal grains. Flammulina velutipes is cultivated in bottles on a sterilized substrate of sawdust and rice bran. Grifola frondosa is cultivated in bottles or plastic bags on sterilized or pasteurized sawdust plus rice or wheat bran. Pleurotus spp. is cultivated on pasteurized or sterilized chopped wheat straw and/or cottonseed hulls filled into plastic bags, bottles, mesh bags, wooden or plastic trays, etc. Pholiota nameko is produced in bottles containing sterilized sawdust and rice bran. Volvariella spp. is cultivated on a variety of pasteurized agricultural wastes.
A common and critically important step in the cultivation of all mushroom types is the inoculation of the substrate with vegetative mycelia of the mushroom being grown. This is typically referred to as spawning, and the inoculum is referred to as mushroom spawn. The ideal mushroom spawn contains high levels of viable mushroom mycelium and sufficient nutrients to maintain viability of the mycelia during storage of the spawn. The spawn should also contain sufficient nutrients to allow growth from the spawn substrate onto the mushroom growing substrate. Specialty mushrooms are typically inoculated with either grain spawn, sawdust spawn, or more rarely, perlite spawn or liquid inoculation methods.
The technology for making grain based mushroom spawn was first taught by Sinden (U.S. Pat. No. 1,869,517) for the cultivation of Agaricus bisporus. Spawn is generally made from sterilized grain that is inoculated with pure cultures of the desired mushroom strain. Mushroom spawn can be prepared by several methods. In one method, dry grain (rye, millet, wheat, sorghum, or other grain), water, CaCO.sub.3, and (optionally) CaSO.sub.4 are placed in suitable containers and capped with lids that allow passage of air and steam but do not allow the passage of microbes that would contaminate the finished product. Containers are subject to steam sterilization for times and temperatures suitable to render the mixtures commercially sterile. Following cooling, the grain mixure is inoculated with a starter culture of the desired mushroom strain, and incubated under permissive conditions to allow complete colonization of the substrate. Containers are shaken at specific intervals to promote even colonization of the mycelium throughout the mixture. Following complete colonization of the hydrated, sterile grain with the mushroom fungus, the spawn can be used immediately to inoculate the mushroom substrate. The mixtures can also be transferred to plastic bags and refrigerated or refrigerated in the production bottle in anticipation of spawning at a future date. Rye grain spawn (the most commonly used) contains about 2.3 wt % nitrogen on a dry weight basis. The moisture content is optimized for the specific mushroom fungus being cultivated.
An alternate method of grain spawn production involves bulk cooking of grain in large kettles. Grain and water mixtures are heated to hydrate the grain. After draining excess water, the hydrated grain is mixed with CaCO.sub.3 and CaSO.sub.4, filled into bottles or heat resistant plastic bags, sterilized, cooled, inoculated with starter cultures of the desired mushroom strain, and incubated to allow colonization of the grain with the mycelium.
Another method of grain spawn production involves placing grain, water, CaCO.sub.3, and CaSO.sub.4 into steam jacketed mixers. Mixtures are cooked, sterilized, cooled, and inoculated in the mixers. The inoculated sterile grain is aseptically transferred to sterile plastic bags that are ventilated to allow passage of air while maintaining sterility. Following mycelial growth, spawn can be shipped to mushroom production facilities with minimal further handling of the product.
Sawdust spawn can be prepared from a variety of ingredients, depending on local availability. A typical formula (Stamets & Chilton, 1983) is to mix four parts of hardwood sawdust with one part rice or wheat bran. The mixture is soaked in water overnight, drained, filled into bottles and autoclaved to render the mixture commercially sterile. The sterile mixture can be inoculated from agar cultures of the desired mushroom fungus, from sawdust or grain spawn, or from a liquid culture. One advantage of sawdust spawn is that the mushroom mycelium grows on a substrate that is chemically and physically similar to the substrate used for cultivation, thus avoiding physiological changes when the spawn is used to inoculate production containers. A disadvantage of sawdust spawn is that it tends to form clumps that make handling of the spawn difficult. Clumping of spawn also results in a heterogeneous distribution of inoculum in the production substrate, causing inconsistencies in mycelial growth. Sawdust spawn typically contains very low levels of nitrogen.
Perlite spawn is based on a formula reported by Fritsche (1978) and first described by Lemke (1971) for spawn on a perlite substrate. The formula is as follows: perlite (1450 g), wheat bran (1650 g), CaSO.sub.4 2H.sub.2 O (200 g), CaCO.sub.3 (50 g), water (6650 ml). The pH after sterilization is 6.2 to 6.4. This formula is calculated to contain 1.10 to 1.34% nitrogen on a dry weight basis (assuming a typical nitrogen content of wheat bran of 2.24 to 2.72%).
Liquid spawn is made from either agar plate cultures or broth cultures of the desired mushroom fungus. Agar or broth cultures are aseptically transferred to sterile water in a sterile blender jar. The cultures are blended briefly to macerate the mycelium, and the resulting mycelial slurry is used to inoculate production units via pipet or syringe. A distinct advantage of the liquid spawn method is that the very large number of mycelial fragments results in very efficient inoculation. A disadvantage of the method is the difficulty in maintaining aseptic conditions during preparation on the inoculum.
Stoller (U.S. Pat. No. 3,828,470) teaches that Agaricus bisporus mushroom mycelium will not grow on feedstuffs such as cottonseed meal, soybean meal, etc., when used alone as an autoclaved substrate. He also teaches Agaricus spawn in which the cereal substrate has been diluted with an inorganic material containing calcium carbonate or an organic flocculating agent. Nitrogen contents are generally low. For example, Stoller's example 16 is estimated to contain about 0.22% nitrogen. Stoller's example 18 is estimated to contain about 0.7% nitrogen. Stoller also teaches that a fine, granular or powdery spawn is preferable to the large, whole grain particles of grain spawn. This is generally due to the number of "points of inoculum" per unit weight of spawn. There is no indication that Stoller's teachings have ever been used for the production of specialty mushroom spawn.
Brini & Sartor (European Patent Application EP 0 700 884 A1) teach a mixture of a water retaining-dispersing agent (e.g., peat), a buffer, a protein containing component (e.g. soybean meal), a growth promoting component (e.g. corn gluten and/or corn starch), and water. The mixture is sterilized, inoculated with the mushroom fungus, and used to spawn compost for the cultivation of Agaricus bisporus mushrooms. The formulation inoculates the mushroom beds and adds protein, while eliminating residual antimicrobial substances and suppressing the growth of antagonistic molds. Moisture contents of the mixtures are typically 54 to 60%, and the formulations typically contain about 9,000 particles per 100 g. Protein contents of the mixtures are 4 to 20 wt % protein. Use of the mixtures as mushroom spawn is asserted to allow the faster growth of the mushroom and prevent the growth of molds. However, routine experimentation has shown that the mixtures taught by Brini & Sartor tend to form clumps, resulting in incomplete sterilization and areas within the mixtures that are not completely colonized by the Agaricus bisporus mycelium. The failure to achieve sterilization results in an economic loss, while a poorly colonized mixture can allow the growth of competitor molds and bacteria in the compost, causing high compost temperatures and reducing mushroom yield.
Romaine (U.S. Pat. No. 4,803,800) teaches production of an Agaricus biporus mushroom casing spawn by encapsulation of nutrients in a hydrogel polymer. Casing spawn is used to inoculate the Agaricus mushroom casing layer rather than the compost. Use of casing spawn speeds fruiting. Specialty mushrooms generally neither require a casing layer not benefit from its use, so this technology is not germane to the present invention. This information is cited here because casing spawn can potentially be used as a substrate spawn. Nitrogen contents in the Romaine casing spawn are generally low. For example, Romaine teaches total nutrient levels of 2 to 6% (wt/vol of formula). Assuming the use of 100% protein as the nutrient source, total nitrogen would be about 0.96 %. Some of Romaine's formulas contain Perlite, vermiculite, soy grits, or similar materials at about 2 to 6% (wt/vol) of the formula as texturizing agents.
Dahlberg & LaPolt (U.S. Pat. No. 5,503,647) teach the development of an Agaricus bisporus mushroom casing spawn prepared from nutritionally inert particles (calcined earth, vermiculite, Perlite, etc) amended with nutrients. Again, specialty mushrooms generally neither require a casing layer not benefit from its use, so this technology is not germane to the present invention. This information is cited here because casing spawn can potentially be used as a substrate spawn. The casing spawn is formulated with low nitrogen contents (generally less than 1%) to allow inoculation of the mushroom casing layer with Agaricus bisporus mycelium without promoting the growth of pests and pathogens. Dahlberg & LaPolt also teach that high levels of proteinaceous ingredients such as soybean fines, etc. are inhibitory to Agaricus bisporus growth. Generally, nitrogen levels above about 2% in a casing spawn formula result in reduced growth of Agaricus bisporus mycelium. This casing spawn formulation is also proposed as a substrate for inoculation of spawn during its preparation.
Some specialty mushroom substrates are also nutritionally amended by the addition of supplements during the spawning process. A mushroom supplement is distinguished from a nutrient ingredient of the substrate in that a supplement is added during the spawning process, after pasteurization. Because the supplements are not sterilized, they are only used in substrates that are pasteuried. In practice, this limits their use to cultivation of Pleurotus, Grifola, Volvariella, and other species that can tolerate a non-sterile substrate. Mushroom supplements are generally made from soybean (i.e., soybean meal, cracked soybeans, etc.), corn (corn gluten), and other agricultural materials. Their addition to a non-sterile mushroom substrate can result in high temperatures that are detrimental to the growth of mushroom mycelia and/or can allow the growth of competitor molds. Supplements are subjected to a variety of treatments to avoid high temperatures and mold growth. These treatments include heat, formaldehyde, fungicides or other mold inhibitory formulations, hydrophobic or hydrophilic coatings, and others (U.S. Pat. Nos. 3,942,969; 4,534,781; 4,617,047; 4,764,199; 5,291,685; 5,427,592). The treatments required to prevent high temperatures and/or mold growth can represent an economic disadvantage and may also raise safety and environmental concerns.